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Download Download Bulletin of Natural Sciences Research DOI: https://doi.org/10.5937/bnsr11-24644 Vol. 11, No. 1, 2021, pp. 1-5. Original Scientific Paper PHARMACEUTICALS AND ENDOCRINE-DISRUPTING COMPOUNDS: ACCUMULATION IN THE TISSUE OF SKADAR LAKE FISH DRAŽANA RADONJIĆ1 1Biology Department, Faculty of Natural Sciences and Mathematics, University of Montenegro, Podgorica, Montenegro ABSTRACT Determining the presence of pharmaceuticals and endocrine-disrupting compounds in fish tissues has been carried out in Montenegro for the first time in Moraca river and Skadar lake. Skadar lake, Montenegro, is the largest of the Balkan lakes and has a surface area which fluctuates seasonally from approximately 370 to 600 km2. During the spring time of 2016, in the time of high water levels of the lake, fish were fetched in triplets and identified. Determining the presence of pharmaceuticals and endocrine-disrupting compounds in fish tissues has been carried out in Montenegro for the first time. Their muscle tissue was separated, grinded, feezed, and then lyophilized. Prepared samples were analyzed with liquid chromatography coupled to tandem mass spectrometry to determine the presence of pharmaceuticals and endocrine-disrupting compounds. Obtained results are under the border of detection, out of 38 tested distributes, only the tissue of Alburnus scoranza showed the presence of triclosan. Keywords: Endocrine-disrupting compounds, Lyophilized, Liquid chromatography coupled to tandem mass spectrometry, Alburnus scoranza, Triclosan. these compounds are lipid soluble and therefore potentially INTRODUCTION bioaccumulative in the environment (Huerta et al., 2013) and fish In recent years, potential risks associated with the release tissue. Moreover, when considering bioaccumulation of PhACs of Endocrine Disrupting Compounds (EDCs) into the aquatic and EDCs in aquatic organisms, one must take other factors into environment have become an increasingly important issue for consideration, such as the different rates of metabolism in environmental regulators. Several investigations have shown that various organisms, how their metabolites accumulate, and the the substances of pharmaceutical origins are often not eliminated uptake and depuration kinetics. during wastewater treament, and are not biodegradable as well Situated along the Montenegro-Albanian border, Lake (Ternes, 1998; Daughton et al., 1999; Zwiener et al., 2004). Due Skadar is the largest lake of the Balkan Peninsula with unique to the conservative nature of physiological processes, chemicals characteristics, wide range of endemic and rare or endagered affect several aquatic species (e.g. algae, invertabrate and fish) in plant and animal species, recognized as a wetland site of a manner similar to their affect on humans due to comparable international significance according to the RAMSAR Convention target molecules (Fent et al., 2006). Many contaminants have (2002). In spite of its importance, inflowing waters from the been detected in wastewater effluents and surface waters in Moraca river and other regional rivers contaminated by the recent years, including pharmaceuticals (PhACs) and (EDCs). industry, municipal and agricultural activities in the area Many of these compounds are not completely removed in the influence the Lake. Therefore, the Lake has been the subject of various physical, chemical, biological and toxicological wastewater treatment plants (WWTPs) and are released to the environment (Gros et al., 2009). EDCs are designed to modify examinations (Beeton & Karaman, 1981; Rastall et al., 2004; physiological or biochemical functions in target organisms but Rakocevic-Nedovic & Hollert, 2005; Stesevic et al., 2007; can have severe consequences when non-target organisms are Perovic et al., 2012; Kastratovic et al., 2014; Vemic et al., 2014; under their exposure. Petrovic et al., 2016, Perovic et al., 2019). In this study, we Better understanding of how these compounds are assessed the level of bioaccumulation of ECDs and PhACs in transferred from water to the biota could help to determine aquatic organisms (fish tissues) Rutilus prespensis (Karaman, potential ecosystem damage associated with the WWTP effluent 1924), Squalius platyceps (Zupancic et al., 2010), Scardinius discharge contaminants. It is generally accepted that substances knezevici (Bianco & Kottelat, 2005), Chondrostoma ohriadunum with octanol-water partition coefficient (log KOW) values higher (Karaman, 1924), Cyprinus carpio (Linnaeus, 1758) and than or equal to 3 have the potential to bioaccumulate in Alburnus scoranza (Bonaparte, 1845). biological tissues. Many PhACs and EDCs have a log KOW < 3, and thus are not expected to bioaccumulate. However, some of * Corresponding author: [email protected] BIOLOGY 1 EXPERIMENTAL for more than a half of the population of Montenegro. Therefore, Area of study and sampling waste waters are seen as the main pollutants of river Moraca. Animals and experimental design The research location belongs to the area of Skadar lake and its tributaries. Locations are marked on the Figure 1. Samples were fetched in triplets using fishnets in spring of 2016, during the time of high water level of the lake, we separated musscle tissues and repeated the process 3 times, grinded them separatekt in a hand blender, put them into special sample boxes and froze them. Two days later, they were transported to the Marine Institute of Montenegro where they were lyophilized. Sample purification optimization Fish homogenate extracts were spiked with a mixture of the target analytes and a comparison was made between three purification techniques: SPE Florisil cartridges (1 g, 6 ml cartridges), Gel permeation chromatography (GPC) in an Agilent 1260 Infinity high pressure liquid chromatography system in tandem with a diode array detector (HPLC-DAD), SPE Oasis HLB (200 mg, 6 ml cartridges) followed by GPC. For the clean- up method based on SPE Florisil, fish extracts were redissolved in 10 ml of acetonitrile prior SPE, which was performed in a Figure 1. Map of Lake Skadar with sampling locations. J.T.Baker® system. Lake Skadar is the largest lake on the Balkan Pninsula. It is Liquid chromatography and mass spectrometry analysis located in karst area on the Montenegro-Albanian border and covers the surface from approximately 370 to 600 km2, The cromatographic tandem mass spectrometry method depending on seasonal fluctations. This area is classified as an applied for the analysis of fish extracts was adapted from Gros et internationally important RAMSAR site (Lake Skadar, Site, al. (2009). Briefly, chromatographic separations were carried out 3YU003, Ramsar Convention 2002) and promoted as a National with a Waters Acquity Ultra-PerformanceTM liquid Park IUCN Category II. In spite of its importance, inflowing chromatography system, using an Acquity HSS T3 column (50 waters from the Moraca River and other regional rivers mm×2.1mm i.d., 1.8_m particle size) for the compounds contaminated by the industry, municipal and agricultural analyzed in positive electrospray ionization (PI) mode and activities in the area affect the lake. River Moraca Acquity BEH C18 column (50 mm×2.1mm i.d., 1.7_m particle (Mediterranean basin), near the town Podgorica, is relatively size) for the ones analyzed under negative electrospray ionization intact but still receives the treatment of waste water discharge, (NI) mode. For the analysis in PI mode, separation conditions produced by 300,000 citizens. River Moraca emerges at the were as follows: solvent (A) methanol, solvent (B) 10mM formic topographic point of 975 m above the creeks which disembogue acid/ammonium format (pH 3.2) at a flow rate of 0.5 mL/min. under the Javorje and Zebalci slopes, with largest of them being The analysis in NI mode was performed by using acetonitrile (A) Javor and Rzav creeks. River Moraca is 113 km long, which and (B) 5mM ammonium acetate/ammonia (ph=8) at a flow rate makes in the longest river in Montenegro. The researched of 0.6 mL/min. The sample volume injected was 5_L in both locations with the coordinates. cases. The UPLC instrument was coupled to a 5500 OTRAP Vukovci 42°20′ 02′′N; 19° 11′ 60′′ E, ˮLeft branch of river hybrid triple quadrupole-linear ion trap mass spectrometer Moraca“ 42°25′84′′ N; 19°13′ 46′′E ˮRight branch of river (Applied Biosystems).; nitrogen collision gas (CAD) medium; Moraca“ 42°27′70′′ N; 19°12′ 32′′ E ˮKraljeva glavica“-Vranjina source gases GS1 and GS2 were set at 60 and 50 psi, 42° 27′ 57′′ N; 19° 12′ 15′′E ˮTanki rt“- Under the bridge 42° 27′ respectively. Besides the monitoring of the SRM transitions, the 90′′ N, 19° 13′ 49′′ E. relative abundance of the two SRM transitions in the sample Throughout decades, this locality has been under the were compared with those in the standards, and the relative influence of industrial waters originating from Kombinat abundances in the samples must be within ±20% of the two SRM Aluminijuma (CAP) and Machine Factory ˮRadoje Dakić“. ratios in the analytical standards. Prepared samples were sent to Therefore, all researches were conductedin order to determine Catalan Institute for Water Research (ICRA)-Gerona in order to the concentration of PCB, aluminium, heavy metals and oil in determine the existence of certain EmS and EDCs in the fish water, land and biota. However, this industry slowly stoped tissues using the (Huerta et al., 2012) and (Huerta et al., 2013) working during the 90s. Podgorica and its surroundings make up method. BIOLOGY 2 NUMERICAL RESULTS approach. Another example is
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